To determine the mechanism by which D‐site‐binding protein (Dbp) regulates rat calvarial osteoprogenitors (OPCs) osteogenic differentiation. α‐Smooth muscle actin (α‐SMA) + rat calvarial OPCs were extracted and purified using immunomagnetic beads. Cells were transduced with Dbp‐lentivirus and divided into Dbp knockdown, Dbp overexpression and vehicle groups. After osteogenic induction for 21 days, Alizarin red staining and alkaline phosphatase (ALP) activity were examined. Expression levels of Runx2, Ocn, Osterix, Bmp4, Kiss1, and GnRH were determined using a quantitative real‐time polymerase chain reaction. The observed changes in Kisspeptin, GnRH, ERα, and Runx2 were further validated via Western blot analysis. Furthermore, E2 and GnRH secretion levels were detected via an enzyme‐linked immunosorbent assay (ELISA). Chromatin immunoprecipitation (ChIP) and luciferase assay were used to assess the effects of Dbp on the Kiss1 gene promoter. The coexpression of Dbp and Kisspeptin or GnRH was also evaluated via immunofluorescence. Following osteogenic induction, Dbp overexpression significantly increased calcium nodule formation and ALP activity, as well as Runx2, Ocn, Osterix, Bmp4, Kiss1, and GnRH messenger RNA expression, while Dbp knockdown presented the opposite results. Western blot analysis and ELISA results showed that Dbp significantly promotes Runx2, E2/ERα, Kisspeptin, and GnRH expression. These findings were confirmed by the ChIP assay, which indicated that the estrogen receptor promotes Kisspeptin expression after binding to the Kiss1 gene promoter, which is regulated by Dbp. Immunofluorescence assay showed that Dbp coexpression with Kisspeptin or GnRH varied depending on Dbp expression levels. Collectively, the circadian transcription factor Dbp promotes α‐SMA + rat calvarial OPCs osteoblastic differentiation through Kiss1/GnRH/E2 signaling pathway loop.

中文翻译：

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昼夜节律转录因子 Dbp 通过 Kiss1/GnRH/E2 信号通路环促进大鼠颅骨骨祖细胞成骨分化。

确定 D 位点结合蛋白 (Dbp) 调节大鼠颅骨骨祖细胞 (OPCs) 成骨分化的机制。使用免疫磁珠提取和纯化α-平滑肌肌动蛋白（α-SMA）+大鼠颅骨OPCs。将细胞用 Dbp-慢病毒转导并分为 Dbp 敲低、Dbp 过表达和载体组。成骨诱导 21 天后，检查茜素红染色和碱性磷酸酶 (ALP) 活性。使用定量实时聚合酶链反应确定 Runx2、Ocn、Osterix、Bmp4、Kiss1 和 GnRH 的表达水平。通过蛋白质印迹分析进一步验证了 Kisspeptin、GnRH、ERα 和 Runx2 中观察到的变化。此外，通过酶联免疫吸附试验（ELISA）检测 E2 和 GnRH 分泌水平。染色质免疫沉淀 (ChIP) 和荧光素酶测定用于评估 Dbp 对 Kiss1 基因启动子的影响。还通过免疫荧光评估了 Dbp 和 Kisspeptin 或 GnRH 的共表达。成骨诱导后，Dbp 过表达显着增加钙结节形成和 ALP 活性，以及​​ Runx2、Ocn、Osterix、Bmp4、Kiss1 和 GnRH 信使 RNA 表达，而 Dbp 敲低则显示相反的结果。Western印迹分析和ELISA结果显示Dbp显着促进Runx2、E2/ERα、Kisspeptin和GnRH的表达。ChIP 分析证实了这些发现，表明雌激素受体在与 Kiss1 基因启动子结合后促进 Kisspeptin 表达，该基因启动子受 Dbp 调节。免疫荧光分析表明，Dbp 与 Kisspeptin 或 GnRH 的共表达因 Dbp 表达水平而异。总的来说，昼夜节律转录因子 Dbp 通过 Kiss1/GnRH/E2 信号通路环促进 α-SMA + 大鼠颅骨 OPCs 成骨细胞分化。